Standardisation of an efficient RNA isolation protocol from the seeds of Momordica charantia var. muricata (Willd.): A Comparative Evaluation

Abstract

Extraction of high-quality total RNA from Momordica charantia var. muricata seeds is difficult due to the presence of polyphenolic and polysaccharide compounds which bind to nucleic acids and thus they co-precipitate and contaminate the RNA samples. The current study aimed to find out a reliable and reproducible RNA extraction method for obtaining good-quality RNA from the seeds of Momordica charantia var. muricata (Willd). The RNA was isolated from seed tissues with four different extraction protocols including the cetyltrimethylammonium bromide (CTAB) method, TRIzol (Invitrogen) method, GenElute™ total RNA purification kit (Sigma-Aldrich) and Plant RNeasy mini kit (Qiagen). The Qiagen RNeasy plant mini kit was the most efficient extraction method providing the highest RNA yield and good quality. The method successfully enhanced the yield of RNA to 923.27 ± 58.64 ng/μl and electrophoresis revealed two distinct bright 28S and 18S rRNA bands and spectrophotometric quantification detected A260/ A280 ratio of 2.04±0.01 within the desired range of 1.8-2. To verify that the RNA quality obtained was appropriate for downstream molecular applications, the isolated RNA samples were converted into cDNA and PCR amplification of the cDNA was performed using a housekeeping gene (Actin 7) and a dormancy-specific gene (DOG1). The current investigation reports on a method for isolating high-quality RNA from M. charantia var. muricata seeds for future dormancy gene expression studies.